The patterns of methylation which ultimately play a role in the global regulation of gene expression in each cell type are formed during development by a complex process involving de novo synthesis in combination with specific demethylation events. In order to understand how these changes are orchestrated, we have developed a general approach for dissecting the cis acting elements and trans acting factor which direct demodification, and have worked out an in vitro system for studying the biochemistry of this reaction. With this information in hand, it should now be possible to specifically manipulate methylation patterns in vivo in order to affect transcription. Using an approach of this nature, for example, one might be able to rescue hemoglobin synthesis in Thallasemia by turning on the silent gamma globin gene. DNA methylation patterns also play an important role in the control of monoallelic expression. We propose to decipher the central cis and trans acting factors which establish differential modification of imprinted genes during gametogenesis and thus pave the way to understanding the molecular pathology of diseases such as Prader-Willi and Angelman Syndromes. Allele specific methylation apparently also operates in the immune system, where it probably controls the activation of immunoglobin genes. By using gene targeting to study this process in vivo, it should be possible to uncover the basic mechanism by which individual lymphocytes are programmed to express a single antibody.